Water-soluble compositions of bioactive lipophilic compounds

ABSTRACT

Water-soluble compositions comprising a lipophilic compound and a solubilizing agent of the general formula: 
     
       
         {X—OOC—[(CH 2 ) n —COO] m } p —Y  (I)  
       
     
     wherein: 
     X is a residue of a hydrophobic moiety, 
     Y is a residue of a hydrophilic moiety, 
     p is 1 or 2, 
     m is 0 or 1, and 
     n is an integer greater than or equal to 0 
     are disclosed. The lipophilic compound is preferably selected from the group consisting of water-insoluble ubiquinones, ubiquinols, vitamins, provitamins, polyene macrolide antibiotics, and mixtures thereof. The hydrophobic moiety is preferably a sterol or a tocopherol and the hydrophilic moiety is preferably a polyalkylene glycol. In some embodiments, the sterol is cholesterol or sitosterol, the tocopherol is α-(+)-tocopherol, the polyalkylene glycol is a polyethylene glycol or its methyl monoether having an average molecular weight between 400 and 1000, p is equal to 1 or 2, m is equal to 0 or 1 and n is an integer between 2 and 18.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of our co-pending U.S.application Ser. No. 09/511,239, filed Feb. 23, 2000 U.S. Pat. No.6,191,172.

FIELD OF THE INVENTION

The present invention relates to water-soluble compositions of bioactivelipophilic compounds, to compounds useful for the preparation of suchcompositions, to methods of preparing such compounds and compositions,and to the use of such compositions as therapeutics and cosmetics.

BACKGROUND OF THE INVENTION

Many bioactive compounds are highly lipophilic (hydrophobic), meaningthat they are soluble in lipids (oils) and some organic solvents, whilebeing substantially insoluble or only sparsely soluble in water. Thelack of solubility of a bioactive compound in aqueous media is animportant factor limiting its therapeutic applications, making difficultan efficient administration of the compound to a patient. Whenadministered in the form of an oil solution or some kind of water and/oroil suspension or emulsion, lipophilic compounds usually show a poorbioavailability, meaning a low concentration and a long build-up time ofthe compound in the systemic circulation. This lack of bioavailabilityis usually independent of the administration route (topical, oral, orparenteral).

Various approaches to overcoming this limitation are known in the priorart. One known approach consists of dissolving a lipophilic compound ina water-miscible organic solvent, such as ethanol or propylene glycol.When such a solution is admixed with blood or gastrointestinal fluids,however, the lipophilic compound usually precipitates as a solid orliquid emulsion, with a resulting low bioavailability. Furthermore, formany lipophilic compounds no organic, water-miscible solvents exist.Another approach consists of incorporating lipophilic compounds intovarious compositions, frequently inhomogeneous, multiphase emulsions,containing oils and solvents in combination with surfactants. Thesecompositions may improve the bioavailability of the compound withoutsignificantly increasing its solubility in aqueous media, but arenormally suitable only for a particular administration form, usually fortopical applications. Such compositions, which may also induce aprotective immune response in mammals, are of little value fortherapeutic uses where administration of the compound by ingestion orinjection is necessary and where an aqueous solution or a water-solublesolid composition is frequently the only acceptable administration form.

Several approaches to preparing homogenous aqueous solutions oflipophilic bioactive compounds are also known in the prior art. Onemethod consists of preparing a derivative or an analog of a lipophiliccompound having a better solubility in water than the original compound.In the simplest case, this derivative may be a water-soluble salt of thecompound, which salt usually retains the original biological activity,but this approach is applicable only to compounds having acidic or basicproperties. If more substantial modifications are introduced into theoriginal compound to improve its solubility, a decrease or even acomplete loss of the original bioactivity of the compound is frequentlyobserved.

Another method of solubilization consists of preparing a water-solublederivative capable of liberating the original bioactive compound underphysiological conditions. Such derivatives, also known as pro-drugs,usually improve bioavailability of the compound and may also ensure atargeted delivery of the compound or its sustained release over a periodof time. However, this approach usually relies on the presence ofcertain functional groups in the original compound, so it is notuniversally applicable. In addition, synthetic methods of improvingsolubility of a compound by chemical modifications are relativelycomplicated and expensive.

Still another approach to solubilization of bioactive lipophiliccompounds relies on formation of water-soluble complexes. An example arecomplexes with amphipathic compounds containing moieties of two opposingsolubility tendencies (lipophilic and hydrophilic). Such compounds areoften soluble both in organic solvents and in water, so that thesolubilization is usually achieved by dissolving a bioactive lipophiliccompound and an amphipathic compound in a suitable water-miscibleorganic solvent and diluting the solution with water. In some cases theorganic solvent is partially or entirely removed from the original orwater-diluted solution by evaporation or lyophilization and theconcentrate reconstituted with a suitable aqueous medium, withoutprecipitation of the water-insoluble lipophilic compound. When theauxiliary organic solvent cannot be completely removed from thecomposition, this solvent must be pharmaceutically acceptable, whichlimits the choice of applicable solvents.

Bioactive lipophilic compounds in need of solubilization belong tovarious therapeutic categories, such as vitamins (e.g., Vitamin E),antibiotics, in particular macrolide polyene antibiotics(amphotericin-B, nystatin, candicidin), free radicals scavengers (e.g.,tocopherols, ubiquinones), immunosuppressants (e.g., cyclosporine), etc.Various approaches to achieve the solubility and improve thebioavailability of these and other lipophilic compounds are known in theprior art, including formation of water-soluble complexes.

U.S. Pat. No. 5,686,110 discloses water-soluble complexes ofwater-insoluble compounds, including amphotericin-B and cyclosporine,with a polyalkylene oxide polymers end-capped with an alkyl or olefinicgroup, which polymers are soluble both in water and in organic solvents.The water-soluble complexes, which are formed only in the presence of anauxiliary organic solvent, are lyophilized and reconstituted with abuffer solution. The reconstituted aqueous solutions show only a limitedstability, depending mostly on the pH of the solution. Furthermore, theuse of methoxypolyethylene glycol polymers of relatively high molecularweight (2,000 to 5,000) as preferred solubilizing agents increases theamount by weight of polymer necessary for solubilization of thebioactive compound.

U.S. Pat. No. 5,798,333 discloses a homogenous, water-solublecomposition (concentrate) of cyclosporine, which can be diluted with anaqueous solution without precipitation of cyclosporine. The concentratecomprises cyclosporine and tocophersolan (polyoxyethanyl-α-tocopherylsuccinate, TPGS) dissolved in a hydrophilic organic solvent, such aspropylene glycol. Solvent-free compositions are not disclosed, as theywould likely be unstable or inhomogeneous.

WO 96/17626 discloses water-soluble compositions of ubiquinonescomprising polyoxyethanyl-cholesteryl sebacate (PCS) as a solubilizingagent. A ubiquinone, in particular Coenzyme Q₁₀, is solubilized bydissolving both Coenzyme Q₁₀ and PCS in tetrahydrofuran at anapproximate molar ratio of 1:3 and diluting this solution with water.The solution is then evaporated to dryness under reduced pressure andreconstituted with a suitable buffer solution.

Coenzyme Q₁₀ (CoQ₁₀) is a natural compound whose therapeutic potentialhas been recently recognized for a number of disorders, includingcongestive heart failure, muscular distrophy, periodontal disease,correction of drug-induced deficiencies, and immune restoration (AIDS,allergies), to name a few. Coenzyme Q₁₀ is also of great interest tocosmetic industry, since it can be included into cosmetic preparationsas agent slowing down natural skin ageing processes. The biologicalactivity of Coenzyme Q₁₀ is believed to be linked to its ability to actas an antioxidant and free radical scavenger protecting integrity ofcell membranes and to offset the inability of diseased cells tomanufacture sufficient energy for cellular repair, by stimulatingmitochondrial respiration and production of ATP. For effectiveness inboth clinical and cosmetic applications, preparations of Coenzyme Q₁₀with high bioavailability and solubility in aqueous media are usuallyrequired.

Even though various methods of improving solubility of lipophiliccompounds, such as Coenzyme Q₁₀, in aqueous media are known in the priorart, they are not equal in terms of simplicity, scope of applicability,stability of the prepared formulations, etc. The present inventionprovides a new method of solubilization of lipophilic compounds, whichis free of many prior art drawbacks and limitations.

SUMMARY OF THE INVENTION

According to one aspect, the present invention provides a water-solublecomposition comprising a lipophilic compound and a solubilizing agent ofthe general formula:

{X—OOC—[(CH₂)_(n)—COO]_(m)}_(p)—Y  (I)

wherein:

X is a residue of a hydrophobic moiety,

Y is a residue of a hydrophilic moiety,

p is 1 or 2,

m is 0 or 1, and

n is an integer greater than or equal to 0.

The lipophilic compound is preferably selected from the group consistingof ubiquinones, ubiquinols, vitamins, provitamins, polyene macrolideantibiotics, and mixtures thereof. The hydrophobic moiety is preferablya sterol or a tocopherol and the hydrophilic moiety is preferably apolyalkylene glycol. In preferred embodiments, the sterol is cholesterolor sitosterol, the tocopherol is α-(+)-tocopherol, the polyalkyleneglycol is a polyethylene glycol or its methyl monoether having anaverage molecular weight between 400 and 1000, p is equal to 1 or 2, mis equal to 0 or 1 and n is an integer between 2 and 18.

According to another aspect of the invention, a water-solublecomposition comprising a bioactive lipophilic compound and asolubilizing agent of the general formula

{X—OOC—[(CH₂)_(n)—COO]_(m)}_(p)—Y

wherein:

p is 1 or 2,

m is 0 or 1, and

n is an integer in the range O≦n≦18

X is a residue of a hydrophobic moiety selected from the groupconsisting of cholesterol, 7-dehydrocholesterol, campesterol,sitosterol, ergosterol, stigmasterol, and α-, β-, γ-, and Δ-tocopherolsand derivatives thereof, Y is a residue of a hydrophilic moiety,selected from the group consisting of polyalcohols, polyethers,polyanions, polycations, polyphosphoric acids, polyamines,polysaccharides, polyhydroxy compounds, polylysines and derivativesthereof, provided that:

when p and m are equal to 1 and the hydrophobic moiety is(+)-α-tocopherol, n is not equal to 2.

According to another aspect, the invention provides a method ofpreparation and purification of a water-soluble composition comprising asolubilizing agent of the general formula (I), in the presence or in theabsence of an auxiliary organic solvent. The solubilization is achievedby dissolving a water-insoluble lipophilic compound and a solubilizingagent in a water-miscible organic solvent, diluting the solution withwater, and removing the organic solvent and optionally a part of waterunder reduced pressure. Alternatively, a lipophilic compound andsolubilizing agent can be admixed directly in a predetermined molarratio and heated to a temperature higher than the their respectivemelting points, to form a water-soluble composition in the form of aclear melt, which can be then diluted with an aqueous solution to adesired concentration. The water-soluble composition may be additionallypurified by dissolving it in a small amount of water, heating thesolution until the composition separates as a clear liquid phase, andremoving the separated composition from the solution while keeping thetemperature of the solution substantially unchanged.

According to still another aspect, the invention provides pharmaceuticalor cosmetic formulations comprising a water-soluble composition of alipophilic compound and a solubilizing agent of formula (I).

According to yet another aspect, the invention provides novelsolubilizing agents of formula (I) and methods of their preparation andpurification.

According to a further embodiment of the invention, a method is providedfor the treatment of a fungal infection in humans or warm-bloodedanimals in need of such treatment, comprising administering to suchhuman or warm-blooded animal, a therapeutically effective amount of awater-soluble composition, comprising a solubilizing agent selected fromthe group consisting of polyoxyethanyl-sitosterol sebacate,polyoxyethanyl-cholesteryl sebacate and polyoxyethanyl-α-tocopherylsebacate, and a macrolide polyene antibiotic, formulated in a weightratio of solubilizing agent to antibiotic of 2:1 to 4:1, in conjunctionwith a pharmaceutically effective carrier or excipient.

According to a yet further embodiment of the invention, a water-solublecomposition is provided, comprising, a solubilizing agent selected fromthe group consisting of polyoxyethanyl-sitosterol sebacate,polyoxyethanyl-cholesteryl sebacate and polyoxyethanyl-α-tocopherylsebacate, and a macrolide polyene antibiotic, formulated in a weightratio of solubilizing agent to antibiotic of 2:1 to 4:1.

According to a still further embodiment of the invention, a method isprovided for preparing a water-soluble composition, comprising the stepsof,

(a) dissolving the antibiotic and the solubilizing agent in awater-miscible organic solvent, in a weight ratio of solubilizing agentto antibiotic of 2:1 to 4:1,

(b) removing from the solution the organic solvent to achieve a desiredconcentration of the water soluble composition,

(c) dissolving the composition in water, and

(d) drying.

According to a yet further embodiment of the invention, a method isprovided for delivery of α-tocopherol to humans or warm-blooded animalsin need thereof, comprising administering to such human or warm-bloodedanimal, an effective amount of a water-soluble form of vitamin E.

PTS is a water-soluble form of vitamin E. We have found that in the bodyof an animal, water-soluble forms of vitamin E, such as PTS, gethydrolysed and are systemically converted back to α-tocopherol, anactive non-toxic form of vitamin E. Accordingly, PTS can itself beadministered to an animal in need thereof, as a means for delivery ofactive non-toxic vitamin E.

According to a yet further embodiment of the invention, a water-solublecomposition is provided, comprising a solubilizing agent selected fromthe group consisting of polyoxyethanyl-sitosterol sebacate,polyoxyethanyl-cholesteryl sebacate and polyoxyethanyl-α-tocopherylsebacate, and a compound having a high content of polyunsaturated fattyacids and derivatives thereof. Derivatives include mono-, di- andtri-glycerides and their aliphatic esters. Examples of such compoundsinclude fish oils, plant oils and other plant extracts. Further examplesof such compounds can be found in the literature in B. Fitch Haumann:Alternate Sources for n-fatty acids in: Inform—International news onfats, oils and related materials (1998) vol. 19 no. 12, p. 1108, and inG. Fernandes et al. Role of omega-3 fatty acids in health and disease.In: Nutrition Research (1993) vol. 13, 19-45, the disclosures of whichare incorporated herein by reference.

According to a still further embodiment of the invention, awater-soluble composition is provided, comprising a solubilizing agentselected from the group consisting of polyoxyethanyl-sitosterolsebacate, polyoxyethanyl-cholesteryl sebacate andpolyoxyethanyl-α-tocopheryl sebacate, and a bioactive lipophiliccompound selected from the group consisting of a terpene and aterpenoid.

We have now solubilized in water α-tocopheryl acetate, a synthetic formof vitamin E, using various solublizing agents including PTS-600 andPTS-400.

The ratio of solubilizing agent to α-tocopheryl-acetate was in the rangeof 2:1 to 5.5:1 w/w. Water-soluble compositions were obtained usingeither the awaiting solvent eg THF method, or the direct admixing methoddescribed above.

Tocotrienols are water-insoluble tocopherol derivatives, useful asantioxidants. We have now made a pegylated form of a tocotrienolpolyoxyethanyl tocotrienyl sebacate (PTrienS-600), which is useful as asolubilizing agent. The method is described below. In particular, seeexamples 31 and 32. It will be appreciated that this invention is alsoapplicable to other tocotrienols such as those described inwww.eastman.com/online publications, Nutriene tocotrienols. Publicationv-24,(1998), the disclosure of which is incorporated herein byreference. We have also dissolved the tocotrienols in water, using asolubilizing agent eg. PTS-600 or a pegylated tocotrienolspolyoxycthanyltocotrienyl sebacate eg (PtrieneS-600.) (see example 32).The ratio of solubilizing agent to tocotreinols is about 5.5:1 w/w. Thewater-soluble compositions are obtained using either the auxiliarysolvent method, or the direct admixing methods described above.

We have also shown that CoQ₁₀ can be water solublized using eitherPTS-400, or pegylated derivatives of tocotrienols, such aspolyoxyethanyltocotrienyl sebacate (PTriensS-600) as solubilizing agent.The ratio of solubilizing agent to CoQ₁₀ was in the range 2.5:1 to3.5:1. The direct admixing method described above was used. See example33.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the toxicity of various solubilizing agents.

FIG. 2 is a graph showing the bioavailability of CoQ₁₀ in variouswater-soluble compositions.

FIG. 3 is a graph showing the effect of PTS-CoQ₁₀ composition oncellular ATP level in NT2 cells.

FIG. 4 is a graph showing the protective effect of PTS-CoQ₁₀ compositionagainst hypoxia in NT2 cells.

FIG. 5 is a graph showing the effect of PTS-CoQ₁₀ and PTS-Vitamin Ecompositions on the viability of hypoxia-treated NT2 cells.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a method of solubilization of substantiallywater-insoluble, bioactive lipophilic compounds, by providing awater-soluble composition comprising the lipophilic compound and asolubilizing agent of the following general formula:

{X—OOC—[(CH₂)_(n)—COO]_(m)}_(p)—Y  (I)

wherein:

X is a residue of a hydrophobic moiety,

Y is a residue of a hydrophilic moiety,

p is 1 or 2,

m is 0 or 1, and

n is an integer greater than or equal to 0.

The hydrophobic moiety of the solubilizing agent is a hydrophobic(lipophilic) molecule having an esterifiable hydroxy group e.g. a sterolor (α-tocopherol, in particular cholesterol, 7-dehydrocholesterol,campesterol, sitosterol, ergosterol, stigmasterol, or an α-, β-, γ-, orΔ-tocopherol. Cholesterol and sitosterol are preferred sterols,sitosterol being particularly preferred. α-(+)-tocopherol andα-(±)-tocopherol are exemplary tocopherols, α-(+)-tocopherol (vitamin E)being preferred. The residue of the hydrophobic moiety is the entirehydrophobic molecule, except for its esterified hydroxy group, such as3-0-hydroxy group of cholesterol or sitosterol or 6-hydroxy group ofo-tocopherol.

The hydrophilic moiety of the solubilizing agent is a hydrophilicmolecule having an esterifiable hydroxy or carboxy group, for example,selected from the group consisting of polyalcohols, polyethers,polyanions, polycations, polyphosphoric acids, polyamines,polysaccharides, polyhydroxy compounds, polylysines, and derivativesthereof. Of those, polyethers are preferred, polyalkylene glycols beingparticularly preferred. The term “polyalkylene glycol” includes polymersof lower alkylene oxides, in particular polymers of ethylene oxide(polyethylene glycols) and propylene oxide (polypropylene glycols),having an esterifiable hydroxy group at least at one end of the polymermolecule, as well as derivatives of such polymers having esterifiablecarboxy groups. The residue of the hydrophilic moiety is the entirehydrophilic molecule, except for its esterified hydroxy or carboxy groupor groups, such as terminal hydroxy groups of a polyethylene glycolmolecule.

Suitable polyethylene glycols may have a free hydroxy group at each endof the polymer molecule, or may have one hydroxy group etherified with alower alkyl, e.g., a methyl group. Also suitable for the practice of theinvention are derivatives of polyethylene glycols having esterifiablecarboxy groups. Polyethylene glycols are commercially available underthe trade name PEG, usually as mixtures of polymers characterized by anaverage molecular weight. Polyethylene glycols having an averagemolecular weight from about 300 to about 5000 are preferred, thosehaving an average molecular weight from about 600 to about 1000 beingparticularly preferred.

Compounds of formula (I) for which m is equal to 1 can be regarded asdiesters of an alkanedioic acid of the general formulaHOOC—(CH₂)_(n)—COOH. For the practice of the present invention,alkanedioic acids with n from 0 to 18 are preferred, those with n from 6to 10 being particularly preferred. Sebacic acid (n=8) is mostparticularly preferred.

Bioactive lipophilic compounds which can be solubilized usingsolubilizing agents of the present invention belong to a variety oftherapeutic and chemical categories and include ubiquinones, ubiquinols,sterols, vitamins, provitamins and macrolide polyene antibiotics.

Preferred ubiquiniones and ubiquinols are those of the formula:

wherein R is

and k is an integer of from 6 to 12. Those with k equal to 10(ubiquinone-50 or Coenzyme Q₁₀ and ubiquinol-50 or reduced Coenzyme Q₁₀,respectively) are particularly preferred.

Cholesterol, sitosterol, ergosterol and 7-dehydrocholesterol arepreferred sterols. Vitamins A, D, E, and K are preferred vitamins andprovitamin A (β-carotene) is a preferred provitamin. Amongst macrolidepolyene antibiotics, amphotericin-B, nystatin, and candicidin arepreferred.

The water-soluble compositions of the present invention contain abioactive lipophilic compound and a solubilizing agent in a molar ratioof approximately 1:1 to 1:5. The molar ratio of about 1:2 is preferred.The upper limit of the molar ratio is not critical, and the solubilizingagent can be used in any excess. This is not desirable, however, asincreasing the amount of the solubilizing agent decreases theconcentration of the active ingredient in the composition and in itsaqueous solutions.

The water-soluble compositions of the present invention can be preparedby two different procedures, either in the presence or in the absence ofan auxiliary organic solvent. In the first case, a lipophilic compoundand a solubilizing agent are first dissolved in a predetermined molarratio in a water-miscible organic solvent and this solution is thendiluted with a predetermined amount of water, without precipitation ofthe lipophilic compound. The organic solvent and water are then removedby evaporation under reduced pressure. A volatile organic solvent isusually removed first, followed by water, in which case the amount ofwater removed from the solution may be controlled, to achieve a desiredconcentration of the water-soluble composition in the remainingconcentrate. Alternatively, both the organic solvent and water areremoved by evaporation, and the waxy residue is reconstituted with asuitable aqueous medium (such as water, physiological saline, or abuffer solution), to provide a clear aqueous solution.

The organic solvent used is in the above procedure should be a goodsolvent for both the lipophilic compound and the solubilizing agent andhas to be miscible with water. If a water-soluble composition is to beused in a pharmaceutical formulation, this solvent should be alsopharmaceutically acceptable, as the removal of the solvent byevaporation may not always be possible. Examples of solvents suitablefor the practice of the invention are tetrahydrofuran, ethanol, ethyleneglycol, propylene glycol, and acetic acid. Solvents with a low boilingpoint, such as tetrahydrofuran, are preferred.

The amount of the organic solvent is not critical, and is equal to orgreater than the minimum amount of solvent necessary to dissolve thegiven amounts of the lipophilic compound and solubilizing agent. Theamount of water used for the dilution is also not critical, and ispreferably between 10 to 25 times the volume of the organic solvent.

An alternative procedure for preparing water-soluble compositionsaccording to the invention consists of preparing first a mixture of alipophilic compound and a solubilizing agent in a predetermined molarratio. This mixture is then heated to a temperature higher than therespective melting points of the lipophilic compound and thesolubilizing agent, for a time necessary to obtain a clear melt, whichprocess can be seen as a dissolution of the lipophilic compound in thesolubilizing agent. The melt so obtained can be reconstituted with apredetermined amount of a suitable aqueous medium, to provide a clearaqueous solution of a desired concentration. This method of preparingwater-soluble compositions of the invention is preferred, as it issimpler and avoids limitations of the procedure that relies on anauxiliary organic solvent, such as the pharmaceutically acceptablecharacter of the solvent required for most applications.

The ability of solubilizing agents of the present invention to dissolvelipophilic compounds in the absence of an auxiliary organic solvent canbe used for preparing water-soluble forms of bioactive compounds, inparticular Coenzyme Q₁₀, without purifying it by crystallization aftersynthesis, thus reducing the overall cost of preparing water-solublecompositions of this compound.

Many compositions of the present invention show a decreasing solubilityin water with increasing temperature of the solution. This provides analternative method of isolation and/or purification of suchcompositions. For the purpose of purification, the composition isdissolved in water at a ratio of the composition to water not exceeding1:2 by volume and the solution is heated, for example in a boiling waterbath, for a time necessary to achieve the separation of thewater-soluble composition as a liquid phase, usually a few minutes. Theoily phase is then separated from the hot solution, while keeping thetemperature of the solution substantially unchanged, as cooling of thesolution would increase the solubility of the composition and result ina reduced yield of the recovery. A speedy separation of the oily phaseto avoid the cooling can be achieved, for example, by centrifugation.

The water-soluble compositions of the present invention have a waxyconsistence and may be difficult to manipulate in this highlyconcentrated form. To make them more easily processable as solids, theymay be combined with a suitable solid, water-soluble additives, such asvitamin C, gelatin, a protein supplement, or a polyethylene glycol. Inthe latter case, a polyethylene glycol having an average molecularweight greater than about 5000 is preferred. The ratio of thecomposition and the additive is not critical, but will be usuallylimited to avoid an unnecessary dilution of the active ingredient by theadditive. Such solid composition are particularly useful for thepreparation of certain dosage forms of bioactive compounds, such astablets or granulates.

The compositions of the present invention show an excellent solubilityin water and allow the preparation of aqueous solutions of almost anyconcentration. As the concentrated solutions can be diluted with anaqueous medium in any proportion and over a wide range of pH conditionswithout precipitation of the lipophilic compound, the solubility of thecompound is maintained under physiological conditions, for example afteran oral or parenteral administration of the composition. This normallyresults in an improved bioavailability of the compound.

The compositions of the present invention and aqueous solutions thereofshow an excellent stability over long periods of time (several months atroom temperature, at least one year when refrigerated, or indefinitelywhen frozen) and over wide ranges of temperature and pH conditions(temperatures from −80° C. to 120° C., pH from 2.0 to 8.0). Aqueoussolutions can be repeatedly frozen and thawed without any perceptibledegradation. The stability under high temperature conditions allows aneasy sterilization of the solutions, without compromising the solubilityof the active ingredient.

The water-soluble compositions of the present invention can be easilyincorporated into pharmaceutical or cosmetic formulations, which arethen charcterized by an improved bioavailability of the lipophilicactive ingredient. Such formulations may further contain additionalactive ingredients and/or a pharmaceutically or cosmetically acceptableadditives or vehicles, including solvents, adjuvants, excipients,sweeteners, fillers, colorants, flavoring agents, lubricants, binders,moisturizing agents, preservatives and mixtures thereof. Theformulations may have a form suitable for a topical (e.g., a cream,lotion, gel, ointment, dermal adhesive patch), oral (e.g., a capsule,tablet, caplet, granulate), or parenteral (e.g., suppository, sterilesolution) administration. Among the acceptable vehicles and solventsthat may be employed for administration by injection are water, mildlyacidified water, Ringer's solution and isotonic sodium chloridesolution.

Among water-soluble compositions and pharmaceutical and cosmeticformulations, those comprising Coenzyme Q₁₀ are of particular interest.Coenzyme Q₁₀ is a natural compound whose therapeutic potential has beenrecently recognized for a number of conditions and disorders related tomitochondrial dysfunctions and/or tissue damage caused by free radicalsand oxidants. These include, but are not limited to: cardiovasculardiseases (e.g., congestive heart failure), muscular disorders (e.g.,muscular dystrophy), mitochondrial encephalomyolopaties (i.g., MELAS,KSS), neurodegenerative disorders (e.g., Alzheimer', Parkinson's,Huntington's diseases), restoration of immune deficiencies caused bydrugs or infections (AIDS, allergies, cancer). Of particular interest isthe clinical use of Coenzyme Q₁₀ to minimize tissue damage resulted fromischemia/reperfusion (e.g., stroke, head trauma, angioplasty, organtransplantation, surgery in general). Another area of interest is theuse of Coenzyme Q₁₀ in therapy as adjuvant, for example for infectiousdiseases, in combination with cholesterol-lowering drugs for thetreatment of hypercholesteremia, or in combination with chemotherapeuticagents in the treatment of cancers. Coenzyme Q₁₀ is also of greatinterest to cosmetic industry, as an agent slowing down natural skinageing processes. Water-soluble compositions comprising a macrolidepolyene antibiotic, such as amphotericin-B, nystatin, or candicidin, areof particular interest for the treatment of fungal infections, includingfungal infections in immunocompromised patients.

Bioactive lipophilic compounds in the form of a water-solublecomposition according to the present invention may be administered to awarm-blooded animal, particularly a human, in need of the prophylaxis ortherapy. The dose of a bioactive lipophilic compound and thecorresponding dose of its water-soluble composition for treating theabove-mentioned diseases or disorders vary upon the manner ofadministration, the age, sex, the body weight of the subject, and thecondition being treated, and will be ultimately decided by the attendingphysician or veterinarian. Such an amount of the bioactive compound inthe form of its water-soluble composition as determined by the attendingphysician or veterinarian is referred to herein as a “therapeuticallyeffective amount”.

The biological activity of Coenzyme Q₁₀ is believed to be linked to itsability to act as an antioxidant and free radical scavenger protectingintegrity of cell membranes and to offset the inability of diseasedcells to manufacture sufficient energy for cellular repair, bystimulating the mitochondrial respiration and production of ATP. Foreffectiveness in both clinical and cosmetic applications, preparationsof Coenzyme Q₁₀ with high bioavailability and solubility in aqueousmedia are normally desirable.

Most pharmaceutical and cosmetic formulations proposed to date forCoenzyme Q₁₀ suffer from a number of drawbacks, such as the use ofcomponents causing an undesirable immune response when administeredparenterally or gastric disorders when administered orally. Someformulations are stable only over a narrow range of pH values and mostare characterized by a poor bioavailability of the active component(Coenzyme Q₁₀). In particular, there are no reliable formulations forintravenous administration of Coenzyme Q₁₀, as there are no formulationsuniversally applicable to topical, oral, and parenteral administrationroutes. The water-soluble compositions of the present inventioncomprising Coenzyme Q₁₀ are free of the above drawbacks and can beincorporated into pharmaceutical formulations suitable for alladministration routes, topical, oral, and parenteral, which are stableand characterized by a good solubility and bioavailability of the activecomponent.

The invention further provides novel solubilizing agents of the generalformula

{X—OOC—[(CH₂)_(n)—COO]_(m)}_(p)—Y  (I)

wherein:

p is 1 or 2,

m is 0 or 1, and

n is an integer in the range of O≦n≦18,

X is a residue of a hydrophobic moiety selected from the groupconsisting of cholesterol, 7-dehydrocholesterol, campesterol,sitosterol, ergosterol, stigmasterol, and α-, β-, γ-, and Δ-tocopherolsand derivatives thereof, Y is a residue of a hydrophilic moiety,selected from the group consisting of polyalcohols, polyethers,polyanions, polycations, polyphosphoric acids, polyamines,polysaccharides, polyhydroxy compounds, polylysines and derivativesthereof,

provided that:

when p and m are both equal to 1 and the hydrophobic moiety isα-(+)-tocopherol, n is not equal 2,

Compounds excluded by the proviso are known in the prior art, inparticular polyoxyethanyl-cholesteryl sebacate (PCS) andpolyoxyethanyl-α-tocopheryl succinate (TPGS). However, PCS was onlydisclosed previously as a solubilizing agent for CoQ₁₀, that is, in ourpreviously published PCT application no. WO96/17626.

The compounds of formula (I) can be prepared by standard methods ofsynthetic organic chemistry, well known to those skilled in the art. Inparticular, compounds where p is equal to 1 or 2 and m is equal to 1 canbe prepared by reacting a compound of the general formula X-OH with acompound of the general formula Z—OC—(CH₂)_(n)—CO—Z, where Z is aleaving group, and further reacting the product so obtained with acompound of the general formula HO—Y—OR, wherein R is hydrogen or analkyl, and X, Y and n are as defined hereinbefore. Halogens, inparticular Cl and Br, are preferred as the leaving group Z. Hydrogen anda lower alkyl (C₁-C₄) are preferred for R.

Many solubilizing agents of formula (I) show a decreasing solubility inwater with the increasing temperature of the solution, which provides aconvenient method of purification of these compounds. The steps andconditions of the purification process are substantially the same asthose discussed above for the purification of water-soluble compositionsof the invention.

Various aspects of the present invention will be further illustrated bythe following non-limiting examples.

EXAMPLES

The following abbreviations are used throughout the Examples:

CoQ₁₀, CoQ₁₀—Coenzyme Q₁₀

PCS—polyoxyethanyl-cholesteryl sebacate

PTS—polyoxyethanyl-α-tocopheryl sebacate

PSS—polyoxyethanyl-sitosteryl sebacate

PTD—polyoxyethanyl-α-tocopheryl dodecanodioate

PTSr—polyoxyethanyl-α-tocopheryl suberate

PTAz—polyoxyethanyl-α-tocopheryl azelaate

TPGS—polyoxyethanyl-α-tocopheryl succinate

A number following one of the above abbreviations (e.g., PCS-600)indicates an average molecular weight of the polyoxyethanyl moiety ofthe compound. A number followed by Me abbreviation (e.g., PTS-750Me)indicates a polyoxyethanyl moiety capped with a methyl group(methoxypolyoxyethanyl).

Examples 1 and 2 illustrate methods of preparation of solubilizingagents of the present invention.

Example 1 Preparation of Polyoxyethanyl-sitosteryl Sebacate (PSS-600)

0.83 g of b-sitosterol (Sigma Chem. Co., product #S-5753, approximately60%) was dissolved in 3 ml of dry toluene at 40° C., followed byaddition of 1.33 mmole of triethylamine (TEA). 1.33 mmole of sebacoylchloride dissolved in 2 ml of dry toluene was than added (dropwise,while stirring, and under anhydrous conditions) to the b-sitosterol-TEAsolution. The reaction was carried out for 10 min at room temperature,at which time 2 mmole of PEG-600 (polyethylene glycol, Sigma Chem. Co.,product # P-3390) and 2.66 mmole of TEA dissolved in 3 ml of dry toluenewere added dropwise to the reaction mixture. The reaction was continuedwith stirring for additional 20 min at room temperature and the reactionmixture was extracted four times with 3 ml each time of saturatedsolution of NaCl. The toluene was removed under reduced pressure leavinga waxy residue. This product was dissolved in 15 ml of water andwater-insoluble materials removed by filtration. The filtrate waslyophilized, yielding 0.8 g of pale-yellow waxy product (PSS-600). Thesame method was used for the preparation of polyoxyethanyl-cholesterylsebacate (PCS-600).

Example 2 Preparation of Polyoxyethanyl-α-tocopheryl Sebacate (PTS-600)

A solution of 1 mmole of α-tocopherol (Sigma Chem. Co., product #T-3251) and 1.33 mmole of TEA in 3 ml of dry toluene was added(dropwise, under anhydrous conditions, while stirring) to 1.33 mmole ofsebacoyl chloride dissolved in 2 ml of dry toluene. The reaction wascarried out for 10 min at room temperature, followed by a dropwiseaddition of 2 mmole of PEG-600 (polyethylene glycol, Sigma, P-3390) and2.66 mmole of TEA dissolved in 3 ml of toluene. The reaction wascontinued for additional 20 min at room temperature with constantstirring. The reaction mixture was extracted four times with 3 ml eachtime of saturated solution of NaCl and toluene evaporated under areduced pressure. The product was dissolved in 5 ml of water and theresidual toluene was further removed by co-evaporation with water undera reduced pressure. The final waxy product (1.15 g) was obtained bylyophilization.

Other solubilizing agents (Table 1) were obtained by linkingpolyethylene glycol (average molecular weight 1000, Sigma Chem. Co.,product # P-3515) or methoxypolyethylene glycol (average molecularweight 750, Sigma Chem. Co., product # M-7018) to (α-tocopherol usingadipoyl, suberoyl, azelaoyl or dodecanedioyl dichlorides. They weresynthesized according to the method of Example 2 and their identitieswere confirmed by mass spectrometry analysis applying MALDI-TOFtechnique.

Example 3 provides molecular characteristics of the synthesizedsolubilizing agents.

Example 3 Molecular Characteristic of Solubilizing Agents Obtained byMALDI-TOF Mass Spectrometry

TABLE 1 Molecular mass of synthesized solubilizing agents. Molecularmass Solubilizing agent m/z PSS-600 1194.3 ± 44 PCS-600 1166.1 ± 44PTS-600 1209.7 ± 44 PTD-600 1237.5 ± 44 PTS-750Me 1355.7 ± 44 PTD-750Me1383.7 ± 44 PTS-1000 1605.9 ± 44 PTSr-600 1181.6 ± 44 PTSr-1000 1578.2 ±44 PTAz-600 1195.8 ± 44 PTA-600 1153.5 ± 44 PTSc-600 1125.7 ± 44 PTS-4001009.7 ± 44

Example 4 illustrates a method of purification of solubilizing agents.

Example 4 Purification of Solubilizing Agents

A solubilizing agent prepared according to Examples 1 or 2 was dissolvedin water at 2:1 v/v ratio. The solution was heated in a boiling waterbath for approximately 2 min, until a visible precipitation occurred.This was followed by a brief centrifugation (at least 2000×g) of the hotmixture to achieve separation of the precipitated product which isinsoluble in hot water. The water phase (supernatant) was removed bydecantation leaving a clear pellet of the product containingapproximately 10% of water.

Examples 5 and 6 illustrate a preparation of water-soluble Coenzyme Q₁₀compositions by a direct admixing of the two components.

Example 5 Direct Preparation of Tocopherol-Based Water-SolubleCompositions of Coenzyme Q₁₀

TABLE 2 Components of tocopherol-based water-soluble Coenzyme Q₁₀compositions. Coenzyme Q₁₀ 0.01 g TPGS 0.035 g  Coenzyme Q₁₀ 0.01 gPTS-600 0.03 g Coenzyme Q₁₀ 0.01 g PTD-600 0.03 g Coenzyme Q₁₀ 0.01 gPTS-750Me 0.03 g Coenzyme Q₁₀ 0.01 g PTD-750Me 0.03 g Coenzyme Q₁₀ 0.01g PTS-1000 0.03 g Coenzyme Q₁₀ 0.01 g PTSr-1000 0.03 g Coenzyme Q₁₀ 0.01g PTSr-600 0.03 g Coenzyme Q₁₀ 0.01 g PTAz-600 0.03 g

Table 2 shows amounts of starting materials used for the preparation ofa-tocopherol-based water-soluble compositions of Coenzyme Q₁₀. In eachcase the two components were admixed together at a predetermined ratioand were heated to a temperature higher than their melting points,typically 60° C.-80° C., until the components melted together and formeda clear, transparent and uniform melt. The optimal ratio of asolubilizing agent to Coenzyme Q₁₀ was found to be 2:1 mol/mol or 3:1w/w. These compositions could be stored in sealed vials for at least 2-3years when refrigerated. They could be reconstituted at any time withwater or physiological solution of saline (0.9%), at any ratio, and theyremained water soluble and stable for several month when refrigerated orfrozen.

Example 6 Direct Preparation of Sterol-Based Water-Soluble Compositionsof Coenzyme Q₁₀

TABLE 3 Components of sterol-based water-soluble Coenzyme Q₁₀compositions Coenzyme Q₁₀ 0.01 g PCS-600 0.03 g Coenzyme Q₁₀ 0.01 gPSS-600 0.03 g

Table 3 shows amounts of starting materials used for the preparation ofsterol-based, water-soluble compositions of Coenzyme Q₁₀. In each casethe two components were admixed at a predetermined ratio (the optimalratio of a solubilizing agent to CoQ10 was found to be 2:1 mol/mol or3:1 w/w) and were heated to a temperature higher than their meltingpoints, typically 60° C.-80° C., until the components melted togetherand formed a clear transparent liquid. The liquid was dissolved in water(at a ratio 2:1 v/v) and the solution was heated in a boiling water bathfor approximately 2 minutes, until clear waxy precipitate was formed.The precipitate could than be separated from the hot solution bycentrifugation at 2000×g. To achieve its full water solubility at roomtemperature, the cycle of heating, centrifugation and cooling to roomtemperature, without removal of supernatant between the cycles, wasrepeated 2 to 3 times. After the final centrifugation, the hotsupernatant was decanted and clear transparent pellet of the product wasrecovered. The compositions could be stored refrigerated in sealed vialsfor at least 2-3 years. Aqueous solutions (in water or saline) of theabove compositions could be prepared and they were stable for severalmonths when refrigerated.

Examples 7, 8 and 9 illustrate preparation of water-soluble Coenzyme Q10compositions in the presence of an auxiliary solvent.

Example 7 Solvent-Based Preparation of Water Soluble Compositions ofCoenzyme Q₁₀

TABLE 4 Components of water-soluble compositions of Coenzyme Q_(10.)Coenzyme Q₁₀ 5 g PCS-600 15 g THF 30 ml H₂O 400 ml Coenzyme Q₁₀ 0.01 gPSS-600 0.03 g THF 0.1 ml H₂O 2.5 ml Coenzyme Q₁₀ 0.3 g PTS-600 1.0 gTHF 4.0 ml H₂O 60.0 ml Coenzyme Q₁₀ 0.01 g PTD-600 0.03 g THF 0.1 ml H₂O2.5 ml Coenzyme Q₁₀ 0.01 g PTS-750Me 0.03 g THF 0.1 ml H₂O 2.5 mlCoenzyme Q₁₀ 0.01 g PTD-750Me 0.03 g THF 0.1 ml H₂O 2.5 ml

Table 4 shows amounts of starting materials used for variouswater-soluble Coenzyme Q₁₀ compositions. In each case Coenzyme Q₁₀ and asolubilizing agent were both dissolved in tetrahydrofuran (THF) and thesolution was added to water with vigorous stirring, while maintainingthe temperature of the mixture close to 0° C. The solvent and a part ofwater were then evaporated under a reduced pressure to obtain a desiredconcentration of Coenzyme Q₁₀, usually 80-100 mg/ml. These compositionscould be stored refrigerated for at least 2-3 years and could bereconstituted with aqueous media (water, saline) to a desired finalconcentration of Coenzyme Q₁₀ in the water-soluble form. Compositionswith higher concentrations of Coenzyme Q₁₀ (up to 200 mg/ml) were alsoobtained by following this procedure.

Example 8 Solvent-Based Preparation of Water-Soluble TPGS-Coenzyme Q₁₀Composition

TABLE 5 Components of water-soluble TPGS-Coenzyme Q₁₀ composition.Coenzyme Q₁₀ 0.01 g TPGS 0.035 g THF 0.1 ml H₂O 2.5 ml

Table 5 shows amounts of starting materials used for water-solubleTPGS-Coenzyme Q₁₀ composition which was prepared according to theprocedure described in Example 7. However, this product did notprecipitated from an aqueous solution after heating. Water and thesolvent could be removed by evaporation under a reduced pressure. Watersolubility and stability of the composition was the same as thosedescribed in Examples 4 and 5.

Example 9 describes a procedure for the preparation of water-solubleubiquinol compositions.

Example 9 Preparation of Water-Soluble Ubiquinol Compositions

TABLE 6 Components of water-soluble ubiquinol compositions. Ubiquinol 500.3 g TPGS 0.9 g Ubiquinol 50 0.3 g TPGS 0.9 g Propylene Glycol usp. 0.9ml Ubiquinol 50 0.1 g Coenzyme Q₁₀ 0.1 g PTS-600 0.5 g Ubiquinol 50 0.1g Coenzyme Q₁₀ 0.1 g Vitamin E 0.001 g PTS-600 0.5 g

Table 6 shows amounts of starting materials used for water-solubleubiquinol (a reduced form of Coenzyme Q₁₀) compositions. Ubiquinol wasfirst prepared by the following method. 0.3 g of Coenzyme Q₁₀ and 0.2 gof zinc dust were suspended in 2.5 ml of glacial acetic acid. Themixture was placed in 50° C. water bath, with occasional shaking, forapproximately 15 min. The reaction mixture was diluted with 2.5 ml ofwater and extracted twice with 5 ml of hexane. The hexane extracts werecombined, washed twice with 2.5 ml of water, dried over anhydrousmagnesium sulfate, evaporated under reduced pressure and finally under ahigh vacuum. The resulting white waxy residue of ubiquinol was combinedwith 0.9 g of TPGS and it was heated to 60° C.-80° C. until the mixturemelted and became clear. Upon reconstitution with water an opaquesolution was formed. This composition remained unchanged for up to 2months when sealed under argon and frozen. The water solubility of thiscomposition could be improved by admixing with propylene glycol at aratio given in the Table 6. The aqueous (water and saline) solutions ofthis composition were stable and could be stored sealed under argon whenfrozen.

Examples 10 and 11 illustrate preparation of water soluble vitamin E andβ-carotene compositions.

Example 10 Preparation of Water-Soluble Formulations of Vitamins

TABLE 7 Components of water-soluble compositions of vitamin E andβ-carotene. Vitamin E 0.10 g PTS-600 0.60 g Vitamin E 0.22 g PCS-6001.00 g THF 2.50 ml H₂O 35.00 ml Vitamin E 0.025 g PTS-600 0.150 g THF0.125 ml H₂O 2.0 ml Provitamin A (β-carotene) 0.01 g PTS-600 0.05 g THF0.20 ml H₂O 3.0 ml

Table 7 shows amounts of starting materials used for variouscompositions prepared by direct admixing method (Example 5) or with theaid of an auxiliary solvent (Example 7). The optimal ratio of asolubilizing agent to Vitamin E was found to be 2:1 mol/mol or 6:1 w/w.The concentrated compositions could be stored refrigerated for up to 2years. The aqueous solutions (in water or saline) were also stable andcould be stored frozen.

Example 11 illustrates methods for isolation, concentration andsterilization of the compositions according to the present invention.

Example 11 Isolation, Concentration and Sterilization of Water-SolubleCompositions

According to the present invention, two processes can be applied for thepreparation of water-soluble compositions of lipophilic compounds: (i) alipophilic compound and a solubilizing agent can either be combineddirectly and melted together, or (ii) the two are first dissolved in awater-miscible auxiliary solvent, the solution mixed with water and boththe solvent and the excess of water are removed by evaporation. Theobtained compositions are soluble in aqueous media at room temperature,but not at temperatures higher than approximately 80° C. Theyprecipitate out of water when heated, but return to the solution uponcooling. This provides a simple, effective and inexpensive method oftheir isolation, concentration and sterilization.

Typically, the aqueous solutions of the compositions (with the exceptionof TPGS-Coenzyme Q₁₀, Example 9) are heated in a boiling water bath forabout 2 min or until visible precipitate is formed. The separation ofthe precipitates from the hot water is accelerated by a rapidcentrifugation at 2000×g. The water phase is decanted and the obtainedwaxy, transparent composition can be stored refrigerated in sealedvials. It remains stable for at least 8 month and can be further dilutedwith aqueous media (water, saline or a buffer). When refrigerated, thesesolutions are stable for at least 3 years. The process ofprecipitation/solubilization is reversible and repeating it severaltimes improves the stability of diluted aqueous solutions of thecomposition. If precipitation of the composition occurs during storage,its solubility can be restored by applying the aboveheating/centrifugation cycle. The compositions are also suitable forinjections, since the described process can be applied for theirsterilization.

Example 12 illustrates the preparation of dry powder compositionscontaining Coenzyme Q₁₀.

Example 12 Preparation of Dry Powder Coenzyme Q₁₀ Compositions

TABLE 8 Components of dry powder Coenzyme Q₁₀ compositions.CoQ10/PCS-600 composition 0.07 g CoQ10/0.21 g PCS-600 containing 40 mgof CoQ10/ml of water BSA (0.25 g/ml of water) 0.25 g CoQ10/PCS-600composition 0.07 g CoQ10/0.21 g PCS-600 containing 40 mg of CoQ10/ml ofwater Gelatin (0.125 g/ml in water) 0.25 g CoQ10/PCS-600 composition0.03 g CoQ10/0.09 g PCS-600 containing 40 mg of CoQ10/ml of waterVitamin C (0.25 g/ml in water) 0.50 g

Table 8 shows amounts of starting materials used for the preparation ofdry powder compositions of Coenzyme Q₁₀. The aqueous solutions of theabove components were combined and lyophilized. The resulting powderedproducts could be pressed into tablets which remained stable andwater-soluble.

Example 13 illustrates the preparation of dry powder compositionscontaining multiple components.

Example 13 Preparation of Compositions Containing Coenzyme Q₁₀, VitaminE and Vitamin C

TABLE 9 Components of water soluble complex compositions. CoenzymeQ10/PTS-600 composition  0.03 g CoQ10/0.09 g PTS-600 (containing 40 mgof CoQ10/ml of water) Vitamin E/PTS-600 composition 0.005 g Vit E/0.025g PTS-600 (containing 20 mg of vitamin E/ml) Vitamin C (0.25 g/ml inwater)  0.75 g

Table 9 shows amounts of starting materials used. The aqueous solutionsof the above components were combined and lyophilized. The resultingpowdered products could be pressed into tablets which remained stableand water-soluble.

Example 14 demonstrates the lack of toxicity of solubilizing agents.

Example 14 In Vitro Toxicity Study

Stock solutions of solubilizing agents (I mg/ml in PBS) were preparedand were added directly to lymphocytic Jurkat and neuroblastoma NT2 cellcultures in the amounts required to achieve a final concentration in themedium of 200, 100 and 50 mg/ml and cell viability was assessed bytrypan blue exclusion assay. The results of assessment are shown in FIG.1.

With the exception of commercially available TPGS, none of thesolubilizing agents of the invention had adverse effects on either cellgrowth or viability in vitro when added to cell cultures atconcentrations up to 200 mg/ml. Also, no toxicity was observed when thecompositions containing CoQ10 and PTS, PCS or PSS were given to SpragueDawley rats at concentrations up to 20 mg/kg body weight, as shown inFIG. 2.

Although no rationale can be provided to explain the toxicity of TPGSobserved in the experimental paradigm used, its usefulness as asolubilizing agent in pharmaceutical formulations should be furtherinvestigated and should be taken with caution.

Example 15 illustrates improved bioavailability of water-soluble formsof CoQ10.

Example 15 Bioavailability of Water-Soluble Forms of Coenzyme Q₁₀

CoQ10, in the form of either water-soluble or oil-soluble compositions,was administered by gavage to 300-350 g male Sprague Dawley rats at adose of 6 mg/kg body weight. Blood samples were collected and CoQ10content was measured in plasma by the HPLC method which we havedeveloped for analysis of CoQ10 in biological samples (Graves et al.,Analysis of CoEnzyme Q10 content in human plasma and other biologicalsamples. In: Free Radicals and Antioxidant Protocols, Ed. D. Armstrong,Humana Press. pp: 353-365, 1998).

Briefly, 0.1 ml sample of plasma or lysed cells (Examples 15 and 16),was mixed with 0.33 ml of 1-propanol, vortexed for 30 sec and left tostand at room temperature for 1 min. 0.86 ml of n-hexane were thanadded, the sample was vigorously vortexed for 30 sec, and centrifuged toachieve a phase separation and to pellet the denatured proteins. Theupper phase which consisted of n-hexane and 1-propanol and containedCoQ₁₀ was collected and evaporated to dryness under argon. The remainingdry residue was redissolved in 60 ml of ethanol and 2 ml of H₂O₂ inorder to convert all CoQ₁₀ to its oxidized form. Sample aliquots wereanalysed by a reverse-phase chromatography on a Supelcosil LC-18-DBcolumn (5 mm particle size, 30 cm×4.0 mm I.D., Supelco) with the mobilephase of ethanol:methanol 80:20 (v/v) at the flow rate of 1 ml/min.Absorbance at 275 and 290 nm was monitored.

A standard calibration curve of CoQ10 and Beckman System Gold Softwarewere utilized for data quantification. The amount of Coenzyme Q₁₀ wascalculated according to measured peak-areas of analysed samples. Thecalibration curve was prepared from a CoQ10 ethanol solution of aconcentration determined spectrophotometrically from its extinctioncoefficient of ε=14,200 at 275 nm. The ethanol solution of CoQ10 can bestored in sealed amber vials at −20° C.

The data presented in FIG. 2 show significantly improved (by 1.5 to 2fold) bioavailability of CoQ10 when given orally as a water solublecompositions in comparison with the oil formulation. Significantly, thekinetics of the uptake is much faster (plasma level peaked at about 3hr) and the maximal plasma levels achieved are also much higher (by 2fold) when CoQ10 is given in the water soluble form.

Example 16 illustrates the ability of cells to internalize thewater-soluble form of CoQ10.

Example 16 Intracellular Uptake of Coenzyme Q₁₀

The following experiments were designed to demonstrate the ability ofhuman keratinocytes to internalize exogenous CoQ10. Human skinkeratinocytes (ATCC CRL-8858), were cultured in SFM (serum free medium,Gibco BRL, cat no. 10724) supplemented with 0.2 ng/ml of EGF (epidermalgrowth factor), 30 mg/ml of bovine pituitary extract and differentconcentrations of CoQ10 which was added directly to the tissue culturemedium in a form of water soluble PCS formulation. The cells were grownfor up to 3 days under these conditions, then harvested and washedextensively. The cells (approximately 1−3×10⁶/per sample) wereresuspended in ddH₂O and were broken by an osmotic shock after afreezing and thawing cycle. CoQ10 was extracted by a propanol/hexanesolvent mixture and its content was measured by the HPLC describedabove.

In order to obtain the mitochondrial fraction, the cells werehomogenized in a buffer containing 250 mM sucrose, 50 mM Tris-HCl pH7.4, 5 mM MgCl₂ and 1 mM EDTA. The homogenates were centrifuged at 800 gfor 10 min at 4° C. The supernatants were collected and were furthercentrifuged at 10,000 g for 10 min at 400. The pellets, representingcrude mitochondrial fractions, were resuspended in 100 ml of ddH₂O,extracted with a propanol/hexane solvent mixture and processed for CoQ10analysis.

TABLE 10 Intracellular uptake of CoQ₁₀ by cultured human keratinocytes.CoQ₁₀ added Total cellular CoQ₁₀ content mg/ml of media ng/10⁶ cells 0-control 10  10 21.1 100 49.6

The data summarized in Table 10 show the intracellular uptake of CoQ10by human keratinocytes from the aqueous environment of tissue culturemedia. During the 3-day experimental period the intracellular content ofCoQ10 increased approximately 2-fold and 5-fold from the mediacontaining 10 mg/ml and 100 mg/ml of CoQ10 resuspended in the PCSformulation, respectively.

TABLE 11 Increased mitochondrial CoQ₁₀ content in keratinocytes. CoQ₁₀added Total mitochondrial mg/ml of Total cellular CoQ₁₀ content contentmedia ng/10⁶ cells ng/10⁶ cells  0 11 1 10 69 9.8

The results shown in Table 11 demonstrate a significant increase ofCoQ10 content in the mitochondrial fraction of cells grown for 3 days inthe presence of 10 mg/ml of CoQ10. It is expected that, in addition toits function as lipid-soluble antioxidant, the elevated CoQ10 content,particularly in the mitochondria, will potentiate the efficiency of theenergy producing respiratory chain which is pivotal for the cellularviability.

Example 17 serves to demonstrate the ability of CoQ10 to increase theefficiency of mitochondrial respiratory chain to produce more ATP.

Example 17 Effects of Coenzyme Q₁₀ on Cellular ATP Content in Human NT2Cells

Human teratocarcinoma NT2 (Stratagene, San Diego, Calif.) were grown inDME medium (GiBco BRL) supplemented with 10% FBS (fetal bovive serum)and 10 mg/ml of CoQ10 which was added directly to the tissue culturemedium in a form of water soluble PTS formulation. The cells wereharvested at 24 hour intervals, resuspended in a buffer consisting of0.02M glycine, 0.05M MgSO₄, 0.004M EDTA, pH 7.4 and aliquoted into 100μL samples.

The ATP content was measured using a luciferin-luciferasebioluminescence assay (Sigma, St. Louis Mo.). Luciferase catalyses thedecarboxylation of luciferin and the hydrolysis of ATP to givepyrophosphate and oxyluciferin. These reactions result in emission oflight at 560 nm. The intensity of the emitted light is proportional tothe ATP concentration. The ATP assay was carried out by mixing 100 μL ofsample with 75 μL of a 0.5 mg/mL solution of luciferase-luciferin.Emitted light was detected using a Beckman LS 3801 scintillationcounter. A standard curve was prepared ranging from 10 to 100 pmols ofATP. ATP levels were expressed as pmols ATP/pg protein.

FIG. 3 shows the effect of PTS-CoQ10 composition on cellular ATP levelin NY2 cells. The ATP level was reproducibly higher in cells that werecultured for 3 days in the presence of water soluble PTS-CoQ10formulation. The data points represent mean values from three separateexperiments +/−SEM.

Example 18 shows protective effects of CoQ10 against hypoxia in humanneuroblastoma NT2 cells.

Example 18 Protection Against Hypoxia

Human teratocarcinoma NT2 (Stratagene, San Diego, Calif.) were seededinto 25 cm² tissue culture flasks (at a density of 0.3×10⁶ cells/ml) andwere grown for 3 days in DME medium (Gibco BRL) supplemented with 10%FBS and in the presence or absence of either 10 mg/ml of CoQ10-PTSformulation added directly to the tissue culture media. The flasks weresealed in an anaerobic chamber containing Gas Pak Plus gas generatorenvelopes and were incubated in the anaerobic conditions at 37° C. for17.5 hours in glucose-free medium. Following the hypoxic treatment thecells were placed again in the complete medium (with and without CoQ10or vitamin E) and were analysed either immediately (time 0) or werecultured for the additional 24 hr under the normoxic conditions. Cellviability was measured by trypan blue exclusion assay. The results areshown in FIG. 4.

The protective effects of CoQ10 against hypoxia in NT2 cells is clearlyevident. Approximately 50% less cells pre-treated for 3 days withPTs-CoO10 died during the 24 hr recovery period from hypoxia as comparedto the cells which did not receive CoQ10.

Example 19 shows that CoQ10 is equally effective in protecting cellsagainst cell death triggered by hypoxia as vitamin E.

Example 19. Comparison of Coenzyme Q10 and Vitamin E Anti-OxidantFunction

Human teratocarcinoma NT2 (Stratagene, San Diego, Calif.) were seededinto 25 cm² tissue culture flasks (at a density of 0.3×10⁶ cells/ml) andwere grown for 3 days in DME medium (Gibco BRL) supplemented with 10%FBS and in the presence or absence of either 10 mg/ml of CoQ10-PISformulation or the same concentration of vitamin E-PTS formulation,which were added directly to the tissue culture media. The flasks weresealed in an anaerobic chamber containing Gas Pak Plus gas generatorenvelopes and were incubated in the anaerobic conditions at 37° C. for17.5 hours in glucose-free medium. Following the hypoxic treatment thecells were placed again in the complete medium (with and without CoQ10or vitamin E) and were analysed either immediately (time 0) or werecultured for the additional 24 hr under the normoxic conditions. Cellviability was measured by trypan blue exclusion assay. The results aresummarized in FIG. 5.

In both experimental paradigms, the CoQ10 and Vitamin E treatments, thepercentage of death during the 24 hr recovery period from hypoxia isreduced by half. This demonstrates that the protective effects of CoQ10against hypoxia-induced cellular damage are comparable to those ofvitamin E, the antioxidant with proven efficacy.

Example 20 demonstrates the protective effects of CoQ10 againstischemia/reperfusion caused tissue damage. Although the animal modelused is the experimental paradigm of human stroke, the data is relevantfor any application where a tissue is damaged as a result ofischemia/reperfusion.

Example 20 Protective Effects of CoQ10 Against Ischemia/Reperfusion

Adult spontaneously hypertensive rats (SHR) weighting 225-250 g, whichreadily develop stroke characteristic lesions after short periods ofvessel occlusion, were used for the middle cerebral artery occlusionmodel (MCAO). The animals were anaesthetized and the right commoncarotid artery was permanently occluded. The skull was exposed and theright middle cerebral artery (MCA) was accessed through a 2 mm burr holein the skull. Under a dissecting microscope a microclip was applied tothe MCA vessel. After a 60 min occlusion period, the clip was removedand the wounds were closed. The body temperature was maintainedthroughout surgery and recovery at 37.5° C. using a rectal thermistorprobe connected to a heating lamp. CoQ10 (6 mg/kg) was administeredintravenously (neck vein) immediately after the removal of the clip. Theeffects of 60 min ischemia were quantitated by tetrazolium salt TTC(2,3,5-triphenyl,2H-tetrazolium chloride)

In order to quantitate the tissue damage, the brains-were cut into 5 mmcoronal sections and were stained overnight with 2% TTC. The colouredformazan was extracted using acetonitrile and its concentration wasmeasured spectrophotometrically at 480 nm. The results as expressed asOD/g of dry brain tissue.

TABLE 12 Protective effects of CoQ10 against ischemia-induced braindamage Untreated animals CoQ₁₀-treated animals Time after Left brainRight brain Time after Left brain Right brain reperfusion hemispherehemisphere reperfusion hemisphere hemisphere Sham 1250.0 +/− 31.8 1239.0+/− 11.9 sham 1250.0 +/− 31.8 1239.0 +/− 11.9 1 day 1208.0 +/− 19.21018.0 +/− 26.0 1 day 1206.0 +/− 50.2 1200.0 +/− 19.2 2 days 1104.0 +/−36.2  898.3 +/− 19.6 2 days 1084.9 +/− 22.2 1060.9 +/− 21.2

Tetrazolium salts are histochemical indicators of mitochondrialrespiratory enzymes and are used to detect tissue infarcts. TTC reactswith intact oxidative enzyme systems, such as succinate and NADHdehydrogenase, accepts electrons and becomes reduced to a coloredformazan which stains tissue red. By contrast, irreversible damagedmitochondria that do not have intact oxidative system cannot reduce TTCand the tissue remains white unstained. Accordingly, the healthier thetissue the higher the OD readings. Compare values obtained for brains ofsham operated control rats (approximately 1250 OD/g of tissue) and forbrains of experimental rats 898.3 OD/g tissue). The protective effectsof CoQ10 against ischemia-induced tissue damage are clearly evident,higher TTC content and, hence, the higher OD readings were obtained frombrains of animals which received CoQ10-treatment postischemically

Example 21 illustrate applicability and suitability of water solubleCoQ10 compositions in cosmetics.

Example 21 Water Soluble CoQ10 Compositions in Cosmetics

TABLE 13 Preparation of cosmetics. CoQ10-PCS-600 Classic MoisturizingCream Final CoQ10 100 mg/ml Oil of Olay concentration 1.5 ml  60 ml0.25% 3.0 ml  60 ml 0.50% CoQ10-PTS-600 Classic Moisturizing Cream FinalCoQ10 100 mg/ml Oil of Olay concentration 1.5 ml  60 ml 0.25% 3.0 ml  60ml 0.50% CoQ10-PTS-600 Dry Skin Moisturizer Final CoQ10 100 mg/ml Pond'sconcentration   5 ml 200 ml 0.25% CoQ10-PTS-600 Moisturizing Body LotionFinal CoQ10 100 mg/ml Vaseline concentration 7.5 ml 300 ml 0.25%CoQ10-PTS-600 Moisturizing Body Lotion Final CoQ10 100 mg/ml Oil of Olayconcentration   5 ml 200 ml 0.25%

Table 13 shows amounts of starting materials used to prepareCoQ10-containing skin products which were combined according to thefollowing procedure. The two components were mixed together at roomtemperature until they formed a uniform composition of a cream in whichCoenzyme Q10 remained water soluble. This was tested by adding water toa 0.2 ml sample of cream (total volume of 1 ml). The samples werevigorously vortexed and centrifuged to separate phases. The aqueousphase remained yellow since it contained dissolved CoQ10. The creamswere stable and no phase separation was observed even after severalmonths at room temperature.

These cosmetics were tested on 20 healthy volunteers over one yearperiod. All reported improved skin conditions, i.e. improved skinelasticity and reduction of wrinkles. None reported an adverse reaction.

Example 22 illustrates preparation of water soluble compositionscontaining polyene macrolide antibiotics.

Example 22 Preparation of Compositions of Anti-Fungal Antibiotics

TABLE 14 Preparation of water-soluble formulations of anti-fungalantibiotics. Candicidin 0.01 g PCS-600 or PTS-600 0.02 g THF/H₂O (8:2,v/v)  0.1 ml H₂O  2.5 ml Nystatin 0.01 g PCS-600 or PTS-600 0.02 gGlacial acetic acid  0.1 ml H₂O  2.5 ml Amphotericin B 0.01 g PCS-600 orPTS-600 0.02 g Methanol/Glacial acetic acid (2:1, v/v)  0.3 ml H₂O  2.5ml

Table 14 shows amounts of starting materials used to preparewater-soluble compositions of antibiotics. Antibiotics and solubilizingagents were dissolved separately in an appropriate solvent or solventmixture. The solutions were combined and diluted with water. Solventsand excess of water were removed by evaporation under a reducedpressure. When glacial acetic acid was used as solvent, theco-evaporation with water was repeated several times. Compositionscontaining 20 mg/ml of antibiotic were obtained by solvent evaporation.They were stable and could be stored frozen for extensive periods oftime (8 months). These compositions could be also isolated, furtherconcentrated and sterilized by the technique described in Example 11.The final products were waxy, clear pellets which could be best storedfrozen, in vials sealed under argon (up to 8 months). They could bereconstituted in aqueous media (water or saline) to desiredconcentrations.

Example 23 illustrates the preparation of water-soluble compositionscontaining polyene macrolide antibiotics.

Example 23 Preparation of Water-Soluble Formulations of AntifungalAntibiotics

TABLE 15 Preparation of water-soluble formulations of anti-fungalantibiotics Amphotericin B, Nystatin  45 mg PCS-600, PTS-600 or PSS-600135-180 mg Methanol/Glacial Acetic Acid (3:1 v/v) 6.0 ml H₂O 4.0 ml

Table 15 shows the amounts of starting materials used for thepreparation of water-soluble compositions of antibiotics. Theantibiotics and solubilizing agents (1:2 to 1:4 w/w ratio) weredissolved directly in the solvent mixture(methanol/glacial acetic acid3:1 v/v). The solvents were removed by evaporation under reducedpressure to obtain waxy residues. The residues were then dissolved inwater, sterilized in boiling water bath for 5 min and were lyophilizedto dryness. The final products were dry powders, which have been storedsealed under argon (for at least 1 year). They were reconstituted inwater, saline or dextrose solution to a desired concentration.Typically, solutions containing 5-30 mg/ml of antibiotics were obtainedby this method. The aqueous solutions of Amphotericicn B were stable forat least 6 months at 4° C.

Examples 24 and 25 illustrate diminished toxicity of amphotericin B andNystatin formulated according to the present invention.

Example 24 In Vitro Haemolysis of Red Blood Cells (RBC)

The haemolysis assay was performed using red blood cells (RBC) isolatedfrom Balb/c mice. The blood was drawn in the presence of heparin (50units/ml), RBC were isolated and washed three times withphosphate-buffered saline (PBS). For the assay, 0.8 ml of 0.1% RBC (v/v)in PBS were mixed with 0.2 ml of PBS buffer containing increasingconcentrations of amphotericin B (final concentrations from 0 to 200jig/ml), formulated with PSS, PCS and PTS or in the generic Fungizone.The mixtures were incubated at 37° C. for 1 h with gentle rotation. Thesamples were spun at 1000×g for 2 min. The content of haemoglobin in thesupernatant was measured by absorbance at 541 nm. The amount ofhaemoglobin, released in the presence of 0.3% Triton X-100 during thesame 1 h at 37° C. incubation period, was taken as a measure of 100%haemolysis.

TABLE 16 Comparison of haemolytic activity of amphotericin B formulatedwith PSS, PCS and PTS with the generic Fungizone. Ampho- Percentage ofLysed Cells tericin (mean value ± SEM)* B PSS-Amph B PCS-Amph B PTS-AmphB (μg/ml) 3:1 w/w 3:1 w/w 4:1 w/w Fungizone 10 1.0 ± 0.3 1.0 ± 0.5  3.0± 0.6 100.0 25 1.0 ± 0.1 1.0 ± 0.4  7.0 ± 3.0 100.0 50 3.0 ± 0.4 7.0 ±2.0 17.0 ± 8.0 100.0 100  3.0 ± 0.6 14.0 ± 3.0 26.0 ± 6.0 100.0 200  1.7± 0.2 22.0 ± 2.0 31.5 ± 7.0 100.0 Note: *mean value ± standard error ofthe mean of three separate experiments, each performed in duplicate.

According to the results presented in Table 16 all three formulations ofamphotericin B, prepared according to the present invention, were farless toxic than the generic Fungizone. However, 10 μg/ml of amphotericinB in Fungizone caused 100% RBCs lysis during 1 hr incubation period, 200μg/ml of the antibiotic formulated with PSS lysed only 1.7% of the cellsduring the same time.

Example 25 In Vitro Haemolysis of Red Blood Cells (RBC)

TABLE 17 Haemolytic activity of Nystatin formulated with PSS, PCS andPTS. Percentage of lysed Cells (mean value ± SEM)* PSS-NystatinPCS-Nystatin PTS-Nystatin Nystatin formulated at a formulated at aformulated at a (μg/ml) ratio 2:1 w/w ratio 2:1 w/w ratio 4:1 w/w TritonX-100  10 2.5 ± 0.8 2.8 ± 0.5 2.0 ± 0.3 100.0  25 2.3 ± 0.7 2.5 ± 0.62.0 ± 0.4 100.0 100 2.2 ± 0.6 2.1 ± 0.7 2.3 ± 0.5 100.0 200 2.1 ± 0.72.3 ± 0.7 2.4 ± 0.5 100.0 Note: *mean value ± standard error of the meanof four separate samples.

As shown in Table 17 all three formulations of nystatin, preparedaccording to the present invention, were non-toxic within theconcentration range of 10 to 200 μg/ml.

Example 26 illustrates in vivo efficacy of the antibiotic formulations.

Example 26 Reduction of C. neoformans Burden in Mouse Brain Following7-Day Treatment with Amphotericin B Formulated Either with PCS or PTSAccording to the Present Invention or With the Generic Fungizone

In vivo efficacy testing of amphotericin B formulations against systemicinfection of murine Cryptococcus neoformans was performed on 8 weeks oldfemale CD1 mice according to the following protocol. All animals wereintravenously inoculated with 1.84×10³ colony forming units (CFU) of C.neoformans. Four days later the mice were subjected to a 7-day treatmentregimen with either the generic Fungizone or amphotericin B-PCS,amphotericin B-PTS and PCS alone (vehicle). The antibiotic formulationswere diluted with 5% dextrose to a final concentration of 250 μg/ml andthe mice received, intravenously, 0.2 ml/dose (equivalent to 2 mg/kgbody weight) of the appropriate antifungal formulation. 24 hr after thelast antifungal treatment, mice were killed and brains were harvested,homogenized and plated on SabDex agar. C. neoformans colonies werecounted after a 3 day incubation at 26° C.

The infection burden of animals treated with the antimycotic agents wascompared to the vehicle treatment.

TABLE 18 C. neoformans infection burden in mice brain after 7-daytreatment with amphotericin B-PCS, amphotericin B-PTS and FungizoneBurden of Number C. neoformans Infection Statistical Antimycotic of mice(mean Log₁₀ reduction analysis formulation (n) of CFU ± SEM)(percentage) (p value) Fungizone  7 5.18 ± 0.58 99.0 <0.0001 Amph B-PCS10 5.37 ± 0.58 98.5 <0.0001 Amph B-PTS 10 5.51 ± 0.41 98.0 0.0003Vehicle  8 7.18 ± 0.30 0 (PCS alone)

The data presented in Table 18 show that both amphotericin Bformulations, prepared according to the present invention, significantlyreduced the C. neoformans burden in brains and that they were equallyeffective as the generic Fungizone.

Example 27 illustrates a release of free (x-tocopherol into bloodstreamfollowing either oral or intravenous administration of PTS.

Example 27 Systemic Release of Vitamin E Following Administration of PTS

PTS, dissolved in water (50 mg/ml), was administered to Sprague Dawleymale rats (300-350 g) either orally or intravenously at a dose of 50mg/kg body weight. Heparinized blood samples were collected at 0, 3, 8and 24 hr following PTS administration. Blood plasma samples wereobtained and analyzed for the content of alpha-tocopherol using an HPLCmethod using a Supercosil LC-18-DB column and a mobile phase ofethanol:methanol (80:20 v/v) at a flow rate of 1 ml/min. Beckman SystemKarat software was used for data quantification.

TABLE 19 Concentration of α-tocopherol in rat blood plasma followingadministration of PTS Time Oral administration Intravenousadministration (hours) (μg/ml) (μg/ml)  0 (control) 6.13 ± 1.18 (n = 3) 6.13 ± 1.18 (n = 3)  3 8.65 ± 1.40 (n = 3) 18.48 ± 2.40 (n = 3)  8 9.33± 2.10 (n = 3) 11.19 ± 3.11 (n = 3) 24 8.31 ± 0.60 (n = 3) 10.59 ± 1.48(n = 3)

Elevated plasma content of α-tocopherol indicates a systemic conversionby the animal body, of PTS to active non-toxic vitamin E.

Example 28 illustrates the ability to dissolve in water different oilswith a high content of polyunsaturated fatty acids (PUFA) such aslinoleic, linolenic, arachidonic eicosapentaenoic (EPA), docosahexaenoic(DHA) and their derivatives.

Example 28 Preparation of Water-Soluble Formulations of Oils ContainingPUFA

Oils were mixed directly with either PTS-600 or PSS-600 at a ratio 1:2to 1:3 w/w. The mixture was then heated to a temperature of 40-50° C. toform a water-soluble composition in the form of a clear melt. Themixture was then diluted with water to form a solution. These watersolutions of PUFA were stable for several months, when refrigerated.

TABLE 20 Preparation of water-soluble formulation of PUFA Flaxseed oil,fish oil 1.0 g PTS-600, PSS-600 2.0-3.0 g

Example 29 illustrates the ability to dissolve in water terpenes andterpenoids including: squalene, geraniol, farnesol, β-carotene,astaxanthin, canthaxanthin, zeaxanthin, cryptoxanthin, lutein andlycopene. Such compounds are useful as antioxidants, colorants andfragrances.

Example 29 Solubilization of Terpenes and Terpenoids

TABLE 21 Preparation of water-soluble formulation of squalene Squalene1.0 g PTS-600, PCS-600, PSS-600 3.0 g

The components are mixed directly with either PTS-600, PCS-600 orPSS-600, at a ratio 1:3 w/w. The mixture was then heated to atemperature of 40-50° C. to form a water-soluble composition in the formof a clear melt. The mixture was then diluted with water to form asolution. The solution is stable for several months, when refrigerated.

TABLE 22 Preparation of water-soluble of astaxanthin Astaxanthin 0.005 gPTS-600 0.020 g THF  0.5 ml H₂O  5.0 ml

Astaxanthin and PTS-600, in a weight ratio of 1:4, are dissolved in awater miscible solvent (THF) and the mixture is diluted with water toform an aqueous solution. The solution was then concentrated under areduced pressure to remove the organic solvent and excess of water. Theconcentrated solution (2 mg/ml) was refrigerated and remained stable forseveral months.

Example 30 illustrates water solubilization of α-tocopheryl acetate.

TABLE 23 Preparation of water-soluble α-tocopheryl acetate α-tocopherylacetate 0.10 g PTS-600 0.51 g THF 1.0 ml H₂O 20.0 ml α-tocopherylacetate 0.10 g PTS-400 0.43 g THF 2.0 ml H₂O 20.0 ml α-tocopherylacetate 0.10 g PTS-400 0.43 g α-tocopheryl acetate 0.10 g PTS-600 0.51 gα-tocopheryl acetate 0.10 g PTrienS-600 0.51 g (polyoxyethanyl--tocotrienyl sebacate)

Table 23 shows amounts of starting materials used for variouswater-soluble formulations of α-tocopheryl acetate. The ratio ofsolubilizing agent to α-tocopheryl acetate was 2:1 mol/mol or 4.5:1 w/wfor PTS-400 and 5.5:1 w/w for PTS-600. Water-soluble formulations wereobtained either with the aid of an auxiliary solvent or by the directadmixing method. The direct admixing was done at an elevated temperatureof hot water bath (80-100° C.). The mixture could be diluted with waterto a desired concentration of α-tocopheryl acetate. The aqueoussolutions obtained by the direct admixing were slightly more opaque thanthe solutions obtained with the aid of auxiliary solvent. The solutionswere stable when refrigerated.

Example 31 illustrates a method for the preparation polyoxyethanyltocotrienyl sebacate (PTrienS).

Example 31 Synthesis of Polyoxyethanyl Tocotrienyl Sebacate(PTrienS-600)

2.15 g of tocotrienols (NuTriene®, Eastmnan Chemical Company) and 0.925ml of triethylamine (TEA) dissolved in 5 ml dry ethyl acetate were addeddropwise to 1.45 ml of sebacoyl chloride, while stirring and underanhydrous conditions. The reaction was carried out for 5 min at roomtemperature, at which time 3 g of PEG-600 (polyethylene glycol, SigmaChem. Co., product # P-3390) and 1.85 ml of TEA, dissolved in 5 ml ofdry ethyl acetate, were added dropwise while cooling in an ice bath. Thereaction was continued with constant stirring for an additional 5 min atroom temperature. The reaction mixture was extracted four times with 5ml (each time) of a saturated solution of NaCl. The reaction productremained soluble in the organic phase from which ethyl acetate wasremoved under reduced pressure, leaving a waxy residue of the product.The product was further dissolved in 50 ml of methanol and themethanol-insoluble by-products were removed by centrifugation, andmethanol was evaporated under reduced pressure. The solid waxy productwas dissolved in 20 ml of water and lyophilized, yielding approximately1.3 g of brownish-waxy solid of PTrienS-600.

Example 32 illustrates water solubilization of tocotrienols.

Example 32 Water Solubilization of Tocotrienols

TABLE 24 Preparation of water-soluble tocotrienols NuTriene 0.1 gPTS-600 0.56 g THF 1.0 ml H₂O 30 ml NuTriene 0.1 g PTS-600 0.56 gNuTriene 0.1 g PTrienS-600 0.56 g

Table 24 shows amounts of starting materials used for variouswater-soluble formulations of tocotrienols. The ratio of solubilizingagent to tocotrienols was about 2:1 mol/mol or 5.5:1 w/w. Water-solubleformulations were obtained either with the aid of an auxiliary solventor by the direct admixing method. The direct admixing was done atelevated temperature of hot water bath (80-100° C.). The mixture wasdiluted with water to a desired concentration of tocotrienols. Theaqueous solutions obtained with PTS were opaque, whereras the solutionobtained with PTrienS was clear. The solutions were stable whenrefrigerated.

Example 33 illustrates water solubilization of Coenzyme Q₁₀ inPTrienS-600 and PTS-400.

Example 33 Preparation of Water-Soluble Formulations of Coenzyme Q₁₀

TABLE 25 Preparation of water-soluble Coenzyme Q₁₀ Coenzyme Q₁₀ 0.012 gPTS-400 0.028 g Coenzyme Q₁₀ 0.010 g PTrienS-600 0.035 g

Table 25 shows amounts of starting materials used to preparewater-soluble formulations of Coenzyme Q₁₀. The ratio of Coenzyme Q₁₀ toPTS-400 was 1:2.5 w/w and to PtrienS-600 1:3.5 w/w. The formulationswere obtained by a direct admixing at elevated temperature of hot waterbath (80-100° C.). The mixture was diluted with water to a desiredconcentration of Coenzyme Q₁₀. The aqueous solutions were stable whenrefrigerated.

What is claimed is:
 1. A method for the treatment of a fungal infectionin humans or warm-blooded animals in need of such treatment, comprisingadministering to such human or warm-blooded animal, a therapeuticallyeffective amount of a water-soluble composition, comprising asolubilizing agent selected from the group consisting ofpolyoxyethanyl-sitosterol sebacate, polyoxyethanyl-cholesteryl sebacateand polyoxyethanyl-α-tocopheryl sebacate, and a macrolide polyeneantibiotic, formulated in a weight ratio of solubilizing agent toantibiotic of 2:1 to 4:1, in conjunction with a pharmaceuticallyeffective carrier or excipient.
 2. A method according to claim 1,wherein the anitibiotic is selected from the group consisting ofamphotericin B and nystatin.
 3. A method according to claim 1, whereinthe antibiotic is amphotericin B, and wherein the weight ratio ofsolubilizing agent to antibiotic is 3:1 to 4:1 w/w.
 4. A methodaccording to claim 1, wherein the antibiotic is nystatin, and whereinthe weight ratio of solubilizing agent to antibiotic is 2:1 to 4:1 w/w.5. A water-soluble composition, comprising a solubilizing agent selectedfrom the group consisting of polyoxyethanyl-sitosterol sebacate,polyoxyethanyl-cholesteryl sebacate and polyoxyethanyl-α-tocopherylsebacate, and a macrolide polyene antibiotic, formulated in a weightratio of solubilizing agent to antibiotic of 2:1 to 4:1.
 6. Acomposition according to claim 5, wherein the antibiotic is selectedfrom the group consisting of amphotericin B and nystatin.
 7. Acomposition according to claim 5, wherein the antibiotic is amphotericinB, and wherein the weight ratio of solubilizing agent to antibiotic is3:1 to 4:1 w/w.
 8. A composition according to claim 5, wherein theantibiotic is nystatin, and wherein the weight ratio of solubilizingagent to antibiotic is 2:1 to 4:1 w/w.
 9. A composition according toclaim 7, wherein the solubilizing agent is polyoxyethanyl-sitosterolsebacate.
 10. A composition according to claim 7, wherein thesolubilizing agent is polyoxyethanyl-cholesteryl sebacate.
 11. Acomposition according to claim 7, wherein the solubilizing agent ispolyoxyethanyl-α-tocopheryl sebacate.
 12. A composition according toclaim 8, wherein the solubilizing agent is polyoxyethanyl-sitosterolsebacate.
 13. A composition according to claim 8, wherein thesolubilizing agent is polyoxyethanyl-cholesteryl sebacate.
 14. Acomposition according to claim 8, wherein the solubilizing agent ispolyoxyethanyl-α-tocopheryl sebacate.
 15. A method for preparing a watersoluble composition according to claim 5, which method comprises thesteps of, (e) dissolving the antibiotic and the solubilizing agent in awater-miscible organic solvent, in a weight ratio of solubilizing agentto antibiotic of 2:1 to 4:1, (f) removing from the solution the organicsolvent to achieve a desired concentration of the water solublecomposition, (g) dissolving the composition in water, and (h) drying.16. A method according to claim 15, wherein the antibiotic isamphotericin B, and wherein the weight ratio of solubilizing agent toantibiotic is 3:1 to 4:1 w/w.
 17. A method according to claim 15,wherein the antibiotic is nystatin, and wherein the weight ratio ofsolubilizing agent to antibiotic is 2:1 to 4:1 w/w.
 18. A methodaccording to claim 15, wherein the solvent is methanol/acetic acid 3:1v/v.
 19. A method according to claim 18, wherein between steps (g) and(h), the composition from step (g) is sterilized.
 20. A method accordingto claim 19, including the additional step of (i) reconstituting thecomposition in water, saline or dextrose solution to a desiredconcentration.